1. Field of the Invention
This invention relates to the chemistry and biology of compounds with immunosuppressive and lymphocyte homing activities and, more specifically, this invention relates to methods and comprises compositions for accelerating lymphocyte homing in a mammal.
2. Description of Related Art
In general, compounds used to suppress the immune response attack certain immune cells. By either removing these cells from the immune system or hampering their ability to respond to chemical messengers, the number of cells participating in any immune response decreases. With fewer cells responding, the immune system cannot mount the same response reaction. The result is immunosuppression.
The use of these compounds follows directly from our understanding of the immune response and the function of immune cells. Numerous publications in the art describe the molecular and cellular aspects of the immune response. Generally, the immune system responds to an antigen first by processing and presenting the antigen through antigen presenting cells (APCs). Over the last decade, intensive research has resulted in a deep and detailed knowledge of this process at the molecular level (1-3). Following the APCs action are the T lymphocytes or T cells. Activated by a process involving antigen presentation by the APC, T cells then initiate the development of a variety of effector immune cells. The activities of phagocytes, natural killer cells, cytotoxic T cells, and B cells and other effector cells each arise from the cytokines secreted from activated T cells. The cytokines, then, are the chemical messengers that trigger the immune response mechanisms that the effector cells perform.
By killing or modifying the cells or messengers of the immune system, certain compounds can be used as treatments and therapies for suppressing the immune response. Our understanding of the immune response has led to two general groups of immunosuppressive compounds, those compounds effecting cytokine action and those directly effecting immune cell metabolism and activity.
In the first group are cyclosporin A (CsA), tacrolimus (TRL) and rapamycin (4-5). CsA, a cyclic peptide, is produced from the Trichoderma polysporum fungus (6-7). TRL, or FK-506, is a macrolide from Streptomyces tsukubaensis (8-10). These compounds cut out the cytokine messengers of the immune response by preventing their synthesis. The immune effector cells, therefore, cannot be recruited to complete the immune response. Rapamycin, on the other hand, blocks the cytokine signal from effecting the immune cells (34).
More specifically, CsA suppresses the immune response by inhibiting production of the cytokine interleukin 2 (IL-2) in antigen-stimulated helper T cells, a subset of T cells. TRL inhibits antigen-induced T cell proliferation by inhibiting IL-2 production in helper T cells. CsA and TRL act by binding to two different proteins (11), called cyclophilin and FKBP respectively. After the binding, both of the CsA/cyclophilin and TRL/FKBP complexes inhibit the phosphatase activity of a protein called calcineurin, which activates nuclear factor (NF-AT) in activated T cells. NF-AT promotes IL-2 gene transcription and thus IL-2 production. However, when the CsA/cyclophilin and TRUFKBP complexes inhibit activation of NF-AT, production of IL-2 is also inhibited.
Since CsA and TRL have almost the same mechanism of action, these drugs also show quite similar side effects, such as renal and liver toxicity (12). Multiple drug therapies with either CsA or TRL, using steroids or other immunosuppressants such as azathioprine and mizoribine (13-14), were widely used in order to reduce the side effects of individual immunosuppressants. However, the similar side effects of CsA and TRL prohibits their use together. New immunosuppressant compounds should not only be highly safe but should also possess a mechanism of action distinct from CsA and TRL so that they can be concomitantly administered.
Compounds from the second group of similarly-acting chemicals each interfere with nucleoside synthesis in the immune cells, arresting their metabolism and their immune activity. The group includes azathioprine (35), mizoribine (36), mycophenolic acid (37), and brequinar sodium (38). These compounds can also result in toxic side effects.
Researchers and clinicians use these compounds in human therapies. Those employing CsA have made great contributions to the prevention of acute rejection in human organ transplantation. Immunosuppressants are also used to treat autoimmune diseases, such as rheumatoid arthritis, and diseases such as psoriasis, atopic dermatitis, bronchial asthma, and pollinosis. However, because of the toxic side effects of the currently used compounds, new, more effective and less toxic methods to suppress the immune response are needed in the art.
The instant invention involves compositions and methods that suppress the immune response in mammals in a novel way. This immunosuppression results from accelerating lymphocyte homing, for example to any of the mesenteric or peripheral lymph tissues or Peyer""s patches. This new activity, accelerated lymphocyte homing immunosuppression (ALH-immunosuppression), can be used in conjunction with other immunosuppressive therapies or compounds while avoiding dangerous or toxic side effects. The present invention provides new and useful methods, therapies, treatments, and compositions wherever immunosuppression is desired or manipulating lymphocyte populations is desired. For example, the invention can be used in therapies or treatments for preventing rejection in organ or cell transplantation, genetically modified cell therapy, ex vivo gene therapy, or other cell therapy methods. Research and development may provide additional or related uses directed to the intestinal immune system and the maintenance or manipulation of intestinal intraepithelial lymphocyte function. Thus, the ALH-immunosuppressive compositions of the invention can be used to direct or redirect lymphocytes within a mammal. Such uses do not necessarily require an immunosuppressive action.
In one embodiment, the invention provides a method of suppressing the immune response by accelerating lymphocyte homing to any of the mesenteric or peripheral lymph tissues or Peyer""s patches. This embodiment can be used to suppress the immune response in a mammal and comprises administering an ALH-immunosuppressive compound. The ALH-immunosuppressive compounds of this invention functionally act by directing lymphocytes to specific locations or lymphoid tissues. This lymphocyte homing activity can be reversible, so that suspending treatment restores normal lymphocyte populations. The compounds may also act to selectively decrease populations of certain lymphocytes in blood or lymph tissue, such as specifically decreasing populations of circulating lymphocytes or spleen lymphocytes.
Structurally, the class of ALH-immunosuppressive compounds derives from myriocin or ISP-1, a natural product of Isaria sinclairii (15). Myriocin is depicted below. 
Numerous homologs, analogs or derivatives of these compounds can be prepared by methods known in the art, such as described in the references, particularly (17), which is specifically incorporated herein by reference. In general, for this invention, the ALH-immunosuppressive compounds can be 2-aminopropane-1,3-diol compounds, according to the following formula: 
wherein R is an optionally substituted straight or branched carbon chain, an optionally substituted aryl, an optionally substituted cycloalkyl or the like;
and R2, R3, R4, and R5 are the same or different and each is a hydrogen, an alkyl, an acyl, or an alkoxycarbonyl, or R4 and R5 may be bonded to form an alkylene chain, which may be substituted by alkyl, aryl, or an alkoxycarbonyl.
Also, for this invention, the ALH-immunosuppressive compounds can be bezene compounds, of the formula: 
wherein W is hydrogen; a straight or branched chain alkyl having 1 to 6 carbon atoms; a straight or branched chain alkenyl having 2 to 6 carbon atoms; a straight or branch chain alkynyl having 2 to 6 carbon atoms; a phenyl, which may be substituted by hydroxy; R4(CH2)n; or a straight or branched chain C1-C6 alkyl substituted by 1 to 3 substituents selected from the group consisting of a halogen, a cycloalkyl, and a phenyl, which may be substituted by hydroxy;
X is hydrogen, a straight-chain alkyl having carbon atoms in the number of p or a straight-chain alkoxy having carbon atoms in the number of (p-1), wherein the straight-chain alkyl having carbon atoms in the number of p and the straight-chain alkoxy having carbon atoms in the number of (p-1) may have 1 to 3 substituents selected from the group consisting of an alkyl, hydroxy, an alkoxy, an acyloxy, amino, an akylamino, an acylamino, oxo, a haloalkyl, a halogen, and a phenyl, which may have a substituent, and wherein the phenyl, which may have a substituent, may have 1 to 3 substituents selected from the group consisting of an alkyl, hydroxy, an alkoxy, an acyl, an acyloxy, amino, an alkylamino, an acylamino, a holalkyl, and a halogen;
Y is hydrogen, an alkyl, hydroxy, an alkoxy, an acyl, an acyloxy, amino, an alkylamino, an acylamino, a haloalkyl, or a halogen;
Z is a single bond or a straight-chain alkylene having carbon atoms in the number of q;
p and q are the same or different and each is an integer of 1 to 20, with the proviso that 6xe2x89xa6p+qxe2x89xa623;
m is 1, 2, or 3;
n is 2 or 3;
R1 and R2 are the same or different and each is hydrogen, an alkyl or an acyl;
R3 is hydrogen, an alkyl or an acyl;
and R4 is hydrogen, an alkyl or an acyl,
where the benzene compounds can be optically active isomers of the above and salts of the compounds.
A disclosure of specific compounds, substituent groups, and variations included in the ALH-immunosuppressive compounds of this invention can be found in U.S. Pat. No. 5,604,229, copending U.S. application Ser. No. 08/801,390, Now U.S. Pat. No. 5,948,820 filed Feb. 20, 1997, and PCT application PCT/JP95/01654, filed Aug. 22, 1995. These documents also describe methods to produce and isolate specific compounds that can be used according to this invention. Also, the references (particularly, 18-20) describe methods for producing these compounds. The entire contents of each of these patent documents and references are specifically incorporated by reference into this disclosure and can be relied on to make or isolate the compounds and practice this invention. The homologs, analogs, or derivatives prepared can be tested, by one skilled in the art, to ensure that they possess ALH-immunosuppressive activity, as described below. Furthermore, the compounds can be prepared or isolated as any of a number of pharmaceutically or physiologically acceptable salts or be prepared as optically active isomers of any of the described compounds.
One preferred structural embodiment of the ALH-immunosuppressive compounds used in the invention is the synthetic product FTY720, 2-amino-2[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride, shown below. 
In other embodiments, the invention provides a method of accelerating lymphocyte homing in a mammal, where a ALH-immunosuppressive composition is used. The composition comprises a 2-aminopropane-1,3-diol compound and/or a benzene compound. In these embodiments, after being administering the composition, the mammal""s immune cells maintain their IL-2-producing ability or that ability is not significantly reduced by the action of the ALH-immunosuppressive composition.
In another embodiment, the invention provides a method for reversibly reducing the number of circulating immune cells in a mammal. These embodiments comprise introducing an ALH-immunosuppressive composition, such as one containing a 2-aminopropane-1,3-diol compound or a homolog or analog thereof, or a benzene compound, or a homolog or analog thereof, to the mammal. A measurable amount of the circulating immune cells are directed to peripheral or mesenteric lymph tissue. In a related aspect, the invention provides a method for manipulating lymphocyte traffic in a mammal comprising administering an ALH-immunosuppressive composition.
By manipulating lymphocyte traffic or reducing circulating lymphocytes, the number of lymphocytes in a particular tissue changes compared to control levels. The examples below indicate changes in lymphocyte numbers for a variety of tissues as well as circulating blood. However, the numbers in the examples do not limit the degree of change in the lymphocyte numbers required. The degree of change can also be any change in lymphocyte numbers that is reasonably attributable to a shift from the control levels as a result of the ALH-immunosuppressive composition or the methods described herein. Alternatively, the degree of change can be those that result in a measurable difference in the immune response of the mammal as determined by any number of assays that one skilled in the art may employ. Also, manipulating lymphocyte traffic or reducing lymphocyte levels in a particular tissue can be determined by following labeled lymphocytes. Changes in the tissue location, the frequency of lymphocyte visits to a tissue, or the numbers of lymphocytes at a specific time period following administration of the labeled lymphocytes, and comparison to control, indicates a manipulation of lymphocyte traffic or a reduction in lymphocyte levels.
In these aspects, an ALH-immunosuppressive composition may also be administered with an antibody, though not necessarily at the same time. Generally, the antibody will be directed against a lymphocyte or an antigen that is involved with the lymphocyte homing process. Numerous antibodies of that type are known in the art. These aspects of the invention can provide, for example, important animal models. The animal models can be used to develop therapies employing the immune system, or its components, or for identifying novel immuno-active compounds or compounds that are involved in the immune response.
In another aspect, the invention relates to a method for identifying the presence or absence of ALH-immunosuppressive activity in a sample, which comprises administering a sample to a mammal and assaying the survival of transplanted tissue or cells in the mammal. By measuring and comparing the ratio of lymphocytes in circulating blood versus lymphocytes in peripheral or mesenteric lymph nodes, before and after treatment, any ALH-immunosuppressive activity can be identified. In these methods, rodents such as rats and mice, may be used and transplanted tissue or cells can be heart, kidney, or skin tissue. Also, for this aspect of the invention, populations of pre-labeled lymphocytes can be introduced into a mammal and detected following administration of a sample. Certain patterns of lymphocyte homing can result when the sample contains ALH-immunosuppressive activity.
In yet another aspect, the invention relates to ALH-immunosuppressive compositions and methods employing these compositions. These compositions comprise a 2-aminopropane-1,3-diol compound, or a homolog or analog thereof, and/or a benzene compound, or a homolog or analog thereof. These compositions can be combined with one or more other immunosuppressive compounds, such as cyclosporin, azathioprine, tacrolimus, mycophenolate mofetil, or analogs or derivatives of these compounds, or steroids, or any other immunosuppressive compound. Because the mechanism of action of the ALH-immunosuppressive activity does not result in similar side effects as in many widely used immunosuppressive compounds, these compositions provide novel synergistic actions, which may allow reduced therapeutic doses.
The ALH-immunosuppressive compositions of this aspect of the invention show superior immunosuppressive effects and are useful themselves, or in methods, for the prevention or treatment of various indications such as immunosuppression in organ, cell, or bone marrow transplantation, various autoimmune diseases or various allergy diseases. Namely, the compositions of the present invention have pharmacological activities such as immunosuppressive activity or antimicrobial activity and therefore are useful for the prevention or treatment of resistance to transplantation or transplantation rejection of organs or tissues (such as heart, kidney, liver, lung, bone marrow, cornea, pancreas, intestinum tenue, limb, muscle, nervus, fatty marrow, duodenum, skin or pancreatic islet cell etc., including xeno-transplantation), graft-versus-host diseases by bone marrow transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, nephrotic syndrome lupus, Hashimoto""s thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes mellitus, type II adult onset diabetes mellitus, uveitis, nephrotic syndrome, steroid-dependent and steroid-resistant nephrosis, palmoplantar pustulosis, allergic encephalomyelitis, glomerulonephritis, etc., and infectious diseases caused by pathogenic microorganisms.
The compositions of the present invention are useful in methods for treating inflammatory, proliferative and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses such as psoriasis, psoriatic arthritis, atopic eczema (atopic dermatitis), contact dermatitis and further eczematous dermatitises, seborrheic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, acne, alopecia areata, eosinophilic fasciitis, and atherosclerosis.
More particularly, the compositions of the present invention are useful in methods for hair revitalizing, such as in the treatment of female or male pattern alopecia, or senile alopecia, by providing epilation prevention, hair germination, and/or a promotion of hair generation and hair growth.
The compositions of the present invention are further useful in methods for the treatment of respiratory diseases, for example, sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, and reversible obstructive airways disease, including conditions such as asthma, including bronchial asthma, infantile asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, particularly chronic or inveterate asthma (for example late asthma and airway hyperreponsiveness), bronchitis and the like.
The compositions of the present invention may also be useful in methods for treating hepatic injury associated with ischemia.
The compositions of the present invention are also indicated in certain methods for treating eye diseases such as conjunctivitis, keratoconjunctivitis, keratitis, vernal conjunctivitis, uveitis associated with Behcet""s disease, herpetic keratitis, conical cornea, dystorphia epithelialis corneae, keratoleukoma, ocular pemphigus, Mooren""s ulcer, scleritis, Graves"" ophthalmopathy, severe intraocular inflammation and the like.
The compositions of the present invention are also useful in methods for preventing or treating inflammation of mucosa or blood vessels (such as leukotriene B4-mediated diseases, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel disease, inflammatory bowel disease (e.g. Crohn""s disease and ulcerative colitis) necrotizing enterocolitis), or intestinal lesions associated with thermal burns.
Further, the compositions of the present invention are also useful in methods for treating or preventing renal diseases including interstitial nephritis, Goodpasture""s syndrome, hemolytic uremic syndrome and diabetic nephropathy; nervous diseases selected from multiple myositis, Guillain-Barre syndrome, Mxc3xa9nixc3xa8re""s disease and radiculopathy; endocrine diseases including hyperthyroidism and Basedow""s disease; hematic diseases including pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis and anerythroplasia; bone diseases including osteoporosis; respiratory diseases including sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin diseases including dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases including arteriosclerosis, aortitis, polyarteritis nodosa and myocardosis; collagen disease including scleroderma, Wegener""s granuloma and Sjxc3x6gren""s syndrome; adiposis; eosinophilic fasciitis; periodontal disease; nephrotic syndrome; hemolytic uremic syndrome; and muscular dystrophy.
Further, the compositions of the present invention are indicated in the treatment of diseases including intestinal inflammations or allergies such as Coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn""s disease or ulcerative colitis; and food related allergic diseases which have symptomatic manifestation remote from the gastrointestinal tract, for example migraine, rhinitis and eczema.
The compositions of the present invention also have liver regenerating activity and/or activity in promoting hypertrophy and hyperplasia of hepatocytes. Therefore, they are useful in methods for the treatment and prevention of hepatic diseases such as immunogenic diseases (e.g. chronic autoimmune liver diseases including autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g. necrosis caused by toxins, viral hepatitis, shock or anoxia), B-virus hepatitis, non-A/non-B hepatitis, and cirrhosis. The compositions of the present invention are also indicated for use as antimicrobial agents, and thus may be used in methods for the treatment of diseases caused by pathogenic microorganisms and the like.
Further, the compositions of the present invention can be used in the prevention or treatment of malignant rheumatoid arthritis, amyloidosis, fulminant hepatitis, Shy-Drager syndrome, pustular psoriasis, Behcet""s disease, systemic lupus erythematosus, endocrine opthalmopathy, progressive systemic sclerosis, mixed connective tissue disease, aortitis syndrome, Wegener""s gramulomatosis, active chronic hepatitis, Evans syndrome, pollinosis, idiopathic hypoparathyroidism, Addison disease (autoimmune adrenalitis), autoimmune orchitis, autoimmune oophoritis, cold hemagglutinin, paroxysmal cold hemoglobinuria, pernicious anemia, adult T cell leukemia, autoimmune atrophic gastritis, lupoid hepatitis, tubulointerstitial nephritis, membranous nephritis, amyotrophic lateral sclerosis, rheumatic fever, postmyocardial infarction syndrome and sympathetic ophthalmitis.
The compositions of the present invention have antifungal effect and are useful as a antifungal agent.
When the compositions are used as pharmaceuticals or in pharmaceutical or pharmacological methods, an effective amount of the 2-aminopropane-1,3-diol compound or a homolog or analog thereof and/or the benzene compound, or a homolog or analog thereof, is generally admixed with carrier, excipient, diluent and so on and formulated into powders, capsules, tablets, injections, topical administration preparations, or the like, for administering to patients. A lyophilized preparation may be produced by a method known in the art.
While the dose of the compounds used in the compositions varies depending on disease, symptom, body weight, sex, age, and so on, they may be administered, for example, to an adult daily by 0.01-10 mg (potency) in a single dose or in several divided doses, for example when suppressing rejection in kidney transplantation.
Moreover, the compositions of the present invention can be used to suppress the immune system, such as suppressing rejection in organ, cell, or bone marrow transplantation. The compositions can comprise combinations with other immunosuppressant(s), steroid(s) (prednisolone, methylprednisolone, dexamethasone, hydrocortisone and the like) or nonsteroidal anti-inflammatory agent(s). Preferred combinations comprise one or more immunosuppressants such as azathiprine, brequinar sodium, deoxyspergualin, mizoribine, mycophenolate 2-morphorinoethyl, cyclosporin, rapamycin, tacrolimus monohydrate, leflunomide, and OKT-3.
Other objects, advantages and applications of this invention will be made apparent by the following detailed description. The description makes reference to preferred and illustrative embodiments of the invention presented in the accompanying drawings.